Light emission as the result of a chemical reaction is known to those skilled in the chemical arts. See Schuster and Schmidt, Chemiluminescence of Organic Compounds, in Advances in Physical Organic Chemistry 18: 187-238 (V. Gold & D. Bethel eds., Academic Press 1982). Additionally, the absorbance or diffusion of light at one or more wavelengths has been applied to the quantifying of bacterial cells in suspension (see Manual of Methods for General Bacteriology 191 (American Society for Microbiology 1981) for the measurement of nucleic acid and protein concentration in solution, id. at 456 and 359 respectively) and as a means of following the purification of various compounds by chromatography and other purification and separation techniques. However, these latter techniques are generally not specific with regard to the identification of a particular compound, such as a protein or nucleic acid species.
The use of chemiluminescent reagents as labeling reagents in analyte-specific immunological assays is known in the art. See e.g., W. Rudolf Seitz, Immunoassay Labels based on Chemiluminescence and Bioluminescence, Clin. Chem. 17:120-126 (1984). The use of acridinium derivatives as specific labeling reagents in such assays has been described in Weeks et al., Acridinium Esters as High Specific Activity Labels in Immunoassays, Clin. Chem. 29:1474-1478 (1983).
Assays employing chemiluminescent labels or "reporter groups" proceed according to a, generalized mechanism. In this mechanism, the light-emitting compound reacts with a second compound which causes the light-emitting compound to enter a transient high energy state. When the excited molecule subsequently returns to a low energy state, a photon is emitted. The reaction may or may not involve additional cofactors or catalysts to facilitate or accelerate the reaction. In a population of such molecules the emitted light can be measured in a light measuring device called a luminometer. The amount of measured light is proportional to the concentration of reacting luminescent compounds in the test sample.
Thus, when the compound is physically associated with an analyte, the amount of light generated is also proportional to the amount of analyte in the sample, so long as any excess or unassociated chemiluminescent reagent has been removed from the sample before reaction and measurement. The compound can be directly bonded to the analyte or can be linked or bonded with a compound which itself is capable of physically associating with the analyte. An example of the latter would be where the chemiluminescent reagent is bonded to an antibody specific for the analyte of interest or to a single-stranded nucleic acid complementary to a nucleic acid whose presence in the test sample is suspected.
Various assay systems for the measurement of more than one specific analyte in a single test sample have been described. In Gorski et al., J. Histochem. and Cytochem. 25:881-887 (1977) a single label, acridine orange, was used as a fluorescent vital dye in mixed lymphocyte cultures. After staining the cultures were monitored at two different wavelengths. Because the dye, which intercalates between the bases of nucleic acids, will emit light in the green region if associated with DNA and in the red region if associated with RNA, it is possible to simultaneously measure total cellular DNA and RNA by monitoring these two wavelength regions.
Various assay systems have been devised employing two or more different radioisotopes each incorporated in one of a binding pair, such as a member of an antibody-antigen pair, a receptor-substrate pair or one of two complementary nucleic acid strands. By using radionuclides emitting different kinds of energy (such as .gamma. radiation and .beta. particle emission) or energies of different intensities it is possible to differentiate between the two radionuclides, and thus between the compounds into which they are incorporated. Scintillation and gamma counters are commercially available which can measure radioactive decay in more than one channel simultaneously.
Thus, in a multi-analyte competition radioimmunoassay (RIA) two or more populations of analyte molecules are labeled with different radioisotopes at a known specific activity (mCi of radioisotope/mmole of analyte). When a test sample is mixed with the labeled analytes, the unlabeled analyte in the test sample will compete with the labeled analyte for binding to an unlabeled specific binding partner. The amount of unlabeled analyte in the test sample is proportional to the decrease in signal as compared to the amount measured without addition of the test sample.
Radioactive assays have obvious disadvantages. Non-radioactive methods for detecting and measuring an analyte in a test sample are known in the art. For example, enzyme-linked immunoassays utilizing biotin and avidin, carbohydrates and lectins have been described, as have assay systems using fluorescent reporter groups such as fluorescein and rhodamine, as well as chemiluminescent reporter groups. Some of these systems also are inherently limited in the sensitivity with which they may detect the analyte of interest due to inherent sensitivity of the label, and/or by the spectral or kinetic characteristics of the particular fluorescent or chemiluminescent compound.
Simultaneous assays of multiple analytes using fluorescent reporter groups having high quantum yields is made more difficult due to the relatively broad spectra and high backgrounds associated with these reagents.
Non-radioactive multiple labeling systems have been reported for the measurement of proteins; Vuori et al., Clin. Chem. 37:2087-2092 (1991), and nucleic acids; Iitia et al., Mol. and Cell. Probes 6:505-512 (1992), in which chelates of fluorescent lanthanides (e.g., europium, samarium and terbium) are coupled to one of a specific binding pair. The unknown components are assayed either through a competition immunoassay or by nucleic acid hybridization, and the fluorescence is measured. The fluorescent lanthanides have narrow emission peaks and the components of the pairs Eu.sup.3+ /Sm.sup.3+ and Eu.sup.3+ /Tb.sup.3+ have emission maxima sufficiently far apart that they may be distinguished from each other. Moreover, the post-excitation fluorescent decay of Eu is relatively long lived, while that of Sm and Tb is extremely short, which provides another way of distinguishing the signals: by measuring the fluorescence of each chelate at different times.
A generalized multiple analyte assay system using acridinium ester derivatives as the reporting group was described in Woodhead et al., PCT Application WO91/00511, which is not admitted to be prior art and which enjoys common ownership with the present application. Khalil et al., PCT Application WO92/12255, describe a solid phase dual analyte immunoassay system employing an acridinium or phenanthridinium derivative as a first chemiluminescent reagent, and a 1,2-dioxetane, which is converted to a chemiluminescent reaction intermediate by alkaline phosphatase or .beta.-galactosidase, as a second chemiluminescent reagent. The acridinium derivative yields a short-lived photon signal upon reaction with a triggering solution such as H.sub.2 O.sub.2. The dioxetane yields a longer-lived signal when triggered by addition of the appropriate enzyme. Each of these reagents can be bonded to one of a specific binding pair and is used in a solid phase sandwich immunoassay. Each signal is measured over a different time period.